In the course of studying inflammatory muscle diseases (polymyositis, dermatomyositis, and related diseases), we have encountered patients with many other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and acid maltase (acid alpha-glucosidase) deficiency (also known as Glycogen Deficiency Type II or GSD II or Pompe Syndrome). For the last several years, we have focused particular attention on GSD II because of its close resemblance to myositis. It is a recessively inherited lysosomal storage disease in which glycogen accumulates in the lysosomes, particularly those in skeletal muscle. When the enzyme, known also as GAA, is completely absent, affected infants are usually sick at birth and die in infancy of heart failure, rarely living longer than a year. Apparently the enzyme is needed in the heart only in infancy since affected individuals with even a small amount of effective enzyme survive without cardiac involvement. Survivors generally develop a progressive proximal myopathy with pulmonary failure secondary to diaphragmatic involvement in later years. The long-term aim of our studies is to prevent and to treat this devastating disease, particularly the adult variety, in which the level of enzyme is only slightly (less than two-fold) below the minimum necessary for a normal life. From quite early in the project, it was driven by the belief that GSDII, being a lysosomal storage disease, should be amenable to enzyme replacement and possibly gene therapy, and since there are well over 30 diseases of abnormal lysosomal storage, our results might have wider application. There are estimated to be several thousand cases of GSD II worldwide, and hundreds in the United States, of which many are fatal in infancy (Pompe Syndrome). Our guiding plan has been to do research directed towards therapy, but without trying to move into areas likely to be covered by pharmaceutical companies. We aimed to develop tools that would advance the development of therapy while at the same time learning new biology or developing techniques that might be applicable to other lysosomal or other enzyme deficiency diseases, especially those in muscle. We have followed lines of research designed to provide answers necessary for the development of optimal therapy for GSD II. Major findings in the previous several years have been summarized in the 2003 annual report . The mouse strains developed in our lab ? particularly the strain that is immunologically toleratent to repeated enzyme injection - have been distributed to many investigators and are now used widely for pre-clinical studies [173]. Our own studies with endogenous expression of the GAA gene in the skeletal muscle {172] and with the intravenous injection of high doses of rhGAA over long periods have shown the remarkable and discouraging finding that unless the enzyme is turned on (controllable transgene) early or the injections are begun early, the stored glycogen in skeletal muscle is only incompletely removed, and clinical recovery of muscle strength is marginal. By contrast, the glycogen stored in cardiac muscle is cleared well. This finding accords with the small clinical experience so far in human infants, in which heart failure is reversed, but skeletal muscle strength has responded little or not at all. This failure of response has now become a major focus of our work. We have discovered that clearance tends to be very good in Type I (slow-twitch) skeletal fibers and very poor in Type II (fast twitch) fibers. In experiments shortly to be published, we have described experiments to determine whether the pathways of trafficking of lysosomal proteins differs between normal and knockout mice and between type I and type II fibers. We have discovered that while the levels of the lysosomal and endosomal markers LAMP-2 and Rab5 do not differ, type II fibers in both normal and knockout mice have dramatically lower levels of three proteins critical for the internalization and trafficking of lysosomal enzymes: the cation-independent mannose-6-phosphate receptor; clathrin; and the adaptor protein complex, AP-2. Furthermore the ratio of the isoforms of AP-2 differed between type I and type II fibers. We have noted that there is striking accumulation of autophagic particles in the type II muscles of affected animals, pointing to a widespread disorder of vacuole trafficking. And that, indeed, is what studies over the past year have confirmed in single fiber studies carried out in collaboration with the Light Imaging Unit of NIAMS. In early studies with single fibers isolated from knockout mice treated by providing transgenic enzyme made endogenously in the liver, it was apparent with confocal immunohistochemical studies that the enzyme reached and cleared glycogen-containing lysosomes in some muscles cells from predominantly type I fibers, but there were other fibers in which there were large presumably glycogen-filled particles that did not contain the enzyme. We have now moved to studying single fibers from mice being treated with enzyme replacement therapy to mimic the clinical situation more closely. We use reagents able to detect not only the administered recombinant enzyme, but also to determine the identity of the various particles: early endosomes (EA1 and Rab5); late endosomes and lysosomes (LAMP); autophagic vacuoles (MAP-LC3). We have solved a number of technical problems to allow these multi-marker experiments to be done on freshly isolated single fibers. In early experiments, it appears that there is a population of large particles whose identity is not completely established in which recombinant enzyme can be detected. This raises the possibilities we have considered either that the enzyme cannot be released; that it cannot reach the substrate; and/or that the conditions are unfavorable for enzyme action. To study these possibilities, we have moved to using fibroblasts from normals or from patients with Pompe syndrome, and, more recently, from normal and knockout mice because the experiments are technically not feasible in single muscle fibers. Dr. Tokiko Fukuda has developed a new technique to allow the determination of pH at several points within single particles in living cells that are doubly labeled with Texas-red dextran and with Oregon-green dextran. What she has found is that there is a population of particles with an unusually high pH. This should reduce the enzymatic activity of the acid alpha-glucosidase and may also diminish its release from the complex in which it has been transported to the particle. Furthermore, labeling studies show that the size, shape, and movement of essentially all of the particles is striking disturbed. Pursuing these disturbances in detail with the aim of reversing them is our major focus in this project. In parallel, we have been doing extensive gene expression studies of the heart and type I and type II muscles from normal and knockout mice at various ages and have uncovered so far that there are abnormal levels of gene expression for many genes in type II muscles in knockout mice compared to normal mice, but few in type I muscles compared to normals. The patterns suggest oxidative damage and autophagy but not apoptosis or atrophy; and certain disturbances of glycolytic metabolism that remain to be understood. In other studies, we have been working for the past year with the Rapoport lab in NIA to attempt to deliver therapeutic levels of GAA across the blood brain barrier to clear CNS neurons of the large quantities of stored glycogen seen in Pompe mice, and which it is presumed will accumulate in the brains of Pompe infants who survive infancy with the help of exogenous enzyme replacement.